Kits for nucleic acid amplification kit using single primer

ABSTRACT

A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3&#39;-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3&#39;-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5&#39; end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.

This is a Continuation of application Ser. No. 08/109,852, filed Aug.20, 1993 now abandoned, which in turn is a file wrapper continuation ofSer. No. 07/734,030, filed Jul. 22, 1991 now abandoned, which in turn isa file wrapper continuation of application Ser. No. 07/399,795, filedAug. 29, 1989 now abandoned, which in turn is a continuation-in-part ofapplication Ser. No. 07/299,282, filed Jan. 19, 1989 now abandoned, thedisclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention.

Nucleic acid hybridization has been employed for investigating theidentity and establishing the presence of nucleic acids. Hybridizationis based on complementary base pairing. When complementary singlestranded nucleic acids are incubated together, the complementary basesequences pair to form double stranded hybrid molecules. The ability ofsingle stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA)to form a hydrogen bonded structure with a complementary nucleic acidsequence has been employed as an analytical tool in molecular biologyresearch. The availability of radioactive nucleoside triphosphates ofhigh specific activity and the ³² P labelling of DNA with T4 kinase hasmade it possible to identify, isolate, and characterize various nucleicacid sequences of biological interest. Nucleic acid hybridization hasgreat potential in diagnosing disease states associated with uniquenucleic acid sequences. These unique nucleic acid sequences may resultfrom genetic or environmental change in DNA by insertions, deletions,point mutations, or by acquiring foreign DNA or RNA by means ofinfection by bacteria, molds, fungi, and viruses. Nucleic acidhybridization has, until now, been employed primarily in academic andindustrial molecular biology laboratories. The application of nucleicacid hybridization as a diagnostic tool in clinical medicine is limitedbecause of the frequently very low concentrations of disease related DNAor RNA present in a patient's body fluid and the unavailability of asufficiently sensitive method of nucleic acid hybridization analysis.

Current methods for detecting specific nucleic acid sequences generallyinvolve immobilization of the target nucleic acid on a solid supportsuch as nitrocellulose paper, cellulose paper, diazotized paper, or anylon membrane. After the target nucleic acid is fixed on the support,the support is contacted with a suitably labelled probe nucleic acid forabout two to forty-eight hours. After the above time period, the solidsupport is washed several times at a controlled temperature to removeunhybridized probe. The support is then dried and the hybridizedmaterial is detected by autoradiography or by spectrometric methods.

When very low concentrations must be detected, the current methods areslow and labor intensive, and nonisotopic labels that are less readilydetected than radiolabels are frequently not suitable. A method forincreasing the sensitivity to permit the use of simple, rapid,nonisotopic, homogeneous or heterogeneous methods for detecting nucleicacid sequences is therefore desirable.

Recently, a method for the enzymatic amplification of specific segmentsof DNA known as the polymerase chain reaction (PCR) method has beendescribed. This in vitro amplification procedure is based on repeatedcycles of denaturation, oligonucleotide primer annealing, and primerextension by thermophilic polymerase, resulting in the exponentialincrease in copies of the region flanked by the primers. The PCRprimers, which anneal to opposite strands of the DNA, are positioned sothat the polymerase catalyzed extension product of one primer can serveas a template strand for the other, leading to the accumulation of adiscrete fragment whose length is defined by the distance between the 5'ends of the oligonucleotide primers.

2. Description of the Prior Art.

A process for amplifying, detecting and/or cloning nucleic acidsequences is disclosed in U.S. Pat. Nos. 4,683,195 and 4,683,202.Sequence polymerization by polymerase chain reaction is described bySaiki, et al., (1986) Science, 230: 1350-1354. A method of making anoligonucleotide is described in European Patent Application No. 0194545A2. Belgian Patent Application No. BE 904402 discloses a mold for makingDNA detection probes. Gene amplification in eukaryotic cells isdisclosed in U.S. Pat. No. 4,656,134.

Langer, et al., Proc, Natl. Acad. Sci. USA, (1981) 78: 6633-6637discloses the enzymatic synthesis of biotin labelled polynucleotides andthe use of these materials as novel nucleic acid affinity probes. Thedetection of viral genomes in cultured cells and paraffin imbeddedtissue sections using biotin labelled hybridization probes is discussedby Brigati, et al., Virology, (1983) 126: 32-50. U.S. Pat. No. 4,486,539discloses the detection of microbial nucleic acids by a one stepsandwich hybridization test. Sensitive tests for malignancies based onDNA detection is described in U.S. Pat. No. 4,490,472. U.S. Pat. No.4,480,040 discloses the sensitive and rapid diagnosis of plant viroiddiseases and viruses employing radioactively labelled DNA that iscomplementary to the viroid or to the nucleic acid of the virus beingdiagnosed. European Patent Application 83106112.2 (Priority U.S. patentapplication Ser. No. 391,440 filed Jun. 23, 1982) teaches modifiedlabelled nucleotides and polynucleotides and methods of preparing,utilizing, and detecting the same. Methods and compositions for thedetection and determination of cellular DNA are disclosed in U.S. Pat.No. 4,423,153. Specific DNA probes in diagnostic microbiology arediscussed in U.S. Pat. No. 4,358,535. A method for detection ofpolymorphic restriction sites and nucleic acid sequences is discussed inEuropean Patent Application No. 0164054 A1. U.S. Pat. No. 4,663,283describes a method of altering double-stranded DNA.

Genomic amplification with transcript sequencing is discussed byStoflet, et al., Science (198) 239:491. Primer-directed enzymaticamplification of DNA with a thermostable DNA polymerase is described bySaiki, et al., Science (1988) 239:487. U.S. Pat. No. 4,724,202 disclosesthe use of non-hybridizable nucleic acids for the detection of nucleicacid hybridization. Bugawan, et al., describe the use of non-radioactiveoligonucleotide probes to analyze enzymatically amplified DNA forprenatal diagnosis and forensic HLA typing.

Detection and isolation of homologous, repeated and amplified nucleicacid sequences is disclosed in U.S. Pat. No. 4,675,283. A singlestranded self-hybridizing nucleic acid probe capable of repeatedlyhybridizing to itself or other nucleic acids to form an amplified entityis described in U.S. patent application Ser. No. 888,058, filed Jul. 22,1986. U.S. Pat. Nos. 4,683,195 and 4,683,202 disclose a homogeneouspolynucleotide displacement assay with digestion of the displaced RNAsingle strand polynucleotide from the reagent complex and amplifyingnucleic acid sequences with treatment of separate complementary strandsof the nucleic acid with two oligonucleotide primers. European PatentApplication No. 0200362 describes a process for amplifying, detecting orcloning nucleic acid sequences and useful in disease diagnosis and inpreparation of transformation vectors. A method for simple analysis ofrelative nucleic acid levels in multiple small samples by cytoplasmicdot hybridization is described in U.S. Pat. No. 4,677,054. Ahybridization method of detecting nucleic acid sequences with a probecontaining a thionucleotide is described in U.S. Pat. No. 4,647,529.

A simple and efficient enzymatic method for covalent attachment of DNAto cellulose and its application for hybridization-restriction analysisand for in vitro synthesis of DNA probes is described in Nucleic AcidsResearch (1986) 14: 9171-9191. Cleavage of single strandedoligonucleotides by Eco RI restriction endonuclease is described inNucleic Acid Research (1987) 15: 709-716.

Exponential Amplification of Recombinant-RNA Hybridization Probes isdescribed by Lizardi, et al. (1988) Bio/Technology 6:1197-1202.Fahrlander, et al., discusses Amplifying DNA Probe Signals: A ChristmasTree Approach in Bio/Technology (1988) 6:1165-1168.

A nucleic acid hybridization assay employing probes cross-linkable totarget sequences is described in U.S. Pat. No. 4,599,303. The methodinvolves the preparation of a specific single stranded ribonucleic acidor deoxyribonucleic acid molecule into which a bifunctionalcross-linking molecule has been covalently incorporated. Theincorporation is such that the cross-linking molecule retains thecapacity to undergo a second reaction with the nucleic acid of thebacterial, viral, or mammalian chromosome, which is the target for theprobe such as to form a covalent cross link. Following cross-linking,the uncrossed link probe is separated from covalently cross-linkedprobe-target complex using one of several procedures which differentiatebetween single stranded probe and double stranded covalently linkedprobe-target complex.

Detection of target sequences in nucleic acids by hybridization usingdiagnostic and contiguous probes for diagnosis of genetic abnormalitydiseases, especially in an automated procedure, is described in EuropeanPatent Application No. 0 185 494A2. In the method a sample is hybridizedwith a probe complementary to a diagnostic portion of the targetsequence (the diagnostic probe) and with a probe complementary to anucleotide sequence contiguous with the diagnostic portion (thecontiguous probe) under conditions wherein the diagnostic probe remainsbound substantially only to the sample nucleic acid containing thetarget sequence. The diagnostic probe and contiguous probe are thencovalently attached to yield a target probe that is complementary to thetarget sequence and the probes which are not attached are removed. In apreferred mode, one of the probes is labeled so that the presence orabsence of the target sequence can then be tested by melting the samplenucleic acid target probe duplex, eluting the dissociated target probe,and testing for the label.

The above method suffers at least one disadvantage in that contiguoussequences are required. To carry out the method, one must identify thediagnostic sequence and the contiguous sequence and create diagnosticand contiguous probes complementary to the above sequences. If thediagnostic and contiguous sequences are not identified precisely, thenthe diagnostic and contiguous probes may not hybridize sufficiently andthe assay specificity and sensitivity can be lost or substantiallydecreased.

SUMMARY OF THE INVENTION

The invention disclosed herein includes methods and reagents forproducing multiple copies of a single stranded polynucleotide wherein asingle polynucleotide primer is employed. As a result, the number ofreagents and assay steps is decreased over known methods.

In one embodiment of the invention, at least one copy of a singlestranded polynucleotide is produced by a method which comprises: (a)forming in the presence of nucleoside triphosphates and templatedependent polynucleotide polymerase along the single strandedpolynucleotide an extension of a polynucleotide primer at least the3'-end of which has at least a 10 base sequence hybridizable with asecond flanking sequence at the 3'-end of the single strandedpolynucleotide, the second flanking sequence being partially or fullycomplementary with at least a 10 base first flanking sequence at the5'-end of the single stranded polynucleotide, (b) dissociating theextended polynucleotide primer and the single stranded polynucleotide,and (c) repeating step (a).

Another aspect of the invention involves a method of producing multiplecopies of a polynucleotide sequence. The method comprises providing incombination (1) a single stranded polynucleotide having thepolynucleotide sequence and being flanked at each end by partially orfully complementary first and second flanking sequences, (2) apolynucleotide primer at least a 10 base portion of which at its 3'-endis hybridizable to that member of the first and second flankingsequences that is at the 3'-end of the single stranded polynucleotide,(3) nucleoside triphosphates, and (4) template dependent polynucleotidepolymerase. The combination is incubated under conditions for eitherwholly or partially sequentially or concomitantly (1) dissociating thesingle stranded polynucleotide from any complementary sequence withwhich it is hybridized, (2) hybridizing the polynucleotide primer withthe flanking sequence at the 3'-end of the single strandedpolynucleotide, (3) extending the polynucleotide primer along the singlestranded polynucleotide to provide a first extended polynucleotideprimer, (4) dissociating the first extended polynucleotide primer andthe single stranded polynucleotide, (5) hybridizing the first extendedpolynucleotide primer with the polynucleotide primer, (6) extending thepolynucleotide primer along the first extended polynucleotide primer toprovide a second extended polynucleotide primer, (7) dissociating thesecond extended polynucleotide primer from the first extendedpolynucleotide primer, and (8) repeating steps (5)-(7).

Another embodiment concerns a method of producing multiple copies of apolynucleotide sequence in a polynucleotide wherein the sequence isflanked at each end by a different member of a pair of flankingsequences that are partially or fully complementary to each other. Themethod comprises (a) combining the polynucleotide with a singlepolynucleotide primer having at least a terminal sequence at its 3'-endat least partially complementary to and hybridizable with at least aportion of the member of the pair of flanking sequences at the 3'-end ofthe polynucleotide sequence, nucleoside triphosphates, and templatedependent polynucleotide polymerase. The combination is incubated underconditions for either wholly or partially sequentially or concomitantly(1) dissociating the polynucleotide sequence from any sequence withwhich it is hybridized to provide a single stranded polynucleotide, (2)hybridizing the polynucleotide primer with the flanking sequence at the3'-end of the single stranded polynucleotide, (3) extending thepolynucleotide primer along the single stranded polynucleotide toprovide an extended polynucleotide primer (4) dissociating the firstextended polynucleotide primer and the single stranded polynucleotide,(5) hybridizing the first extended polynucleotide primer with thepolynucleotide primer, (6) extending the polynucleotide primer along thefirst extended polynucleotide primer to provide a second extendedpolynucleotide primer, (7) dissociating the second extended primer fromthe first extended polynucleotide primer, and (8) repeating steps(5)-(7) above.

The above methods have application in facilitating the determination ofthe presence of a polynucleotide analyte in a sample suspected ofcontaining such polynucleotide analyte. The target polynucleotidesequence can be DNA or RNA.

In one embodiment of this aspect of the invention the method comprises(a) forming as a result of the presence of an analyte a single strandedpolynucleotide flanked by complementary first and second flankingsequences, (b) forming multiple copies of the single strandedpolynucleotide and the flanking sequences, and (c) detecting the singlestranded polynucleotide. Any method for detecting specific nucleic acidsor polynucleotides can be used. Multiple copies of the single strandedpolynucleotide can be made by, for example, any of the aforementionedmethods.

In one particular method for determining the presence of apolynucleotide analyte in a sample suspected of containing the analyte,a single stranded polynucleotide flanked by at least 80% complementaryfirst and second flanking sequences, each comprised of at least 15bases, is formed as a result of the presence of the analyte. In thepresence of nucleoside triphosphates and template dependentpolynucleotide polymerase an extension is formed of a polynucleotideprimer at least the 3'-end of which can hybridize with the secondsequence at the 3'-end of the single stranded polynucleotide. Extendedpolynucleotide primer containing a sequence identical to and/orcomplementary with the single stranded polynucleotide is then detected,the presence thereof indicating the presence of the polynucleotideanalyte.

Another aspect of the present invention concerns an analytical methodwhich comprises contacting a sample suspected of containing the analytewith first and second polynucleotide probes. The first probe comprises asequence at its 3'-end complementary to a first portion of one strand ofthe analyte and a first flanking sequence. The second probe comprises asequence at its 5'-end complementary to a second portion of the samestrand of the analyte and a second flanking sequence. The first andsecond flanking sequences are partially or fully complementary. Thecontact is carried out under conditions for binding of the first andsecond probes with the analyte. Conditions are provided for ligating thefirst and second polynucleotide probes to one another only when bound tothe analyte. An extension of a polynucleotide primer at least the 3'-endof which can hybridize with the flanking sequence is formed in thepresence of nucleoside triphosphates and template dependentpolynucleotide polymerase. Extended polynucleotide primer containing asequence complementary to the first probe is detected, the presencethereof indicating the presence of the analyte. Another aspect of thepresent invention involves a method for detecting the presence of apolynucleotide analyte in a sample suspected of containing the analyte.The method comprises the steps of (a) ligating third and fourthsequences of a polynucleotide probe, the third sequence having a free3'-end and the fourth sequence having a free 5'-end and each beinghybridizable to separate portions of the analyte and ligatable, orcapable of being rendered ligatable, only in the presence of theanalyte, the polynucleotide probe further comprising first and secondflanking sequences, wherein the first sequence is 5' of the thirdsequence, the second sequence is 5' of the first sequence and the fourthsequence is 5' of the second sequence, (b) forming in the presence ofnucleoside triphosphates and template dependent polynucleotidepolymerase an extension of a polynucleotide primer at least the 3'-endof which can hybridize with the second flanking sequence of thepolynucleotide probe, and detecting extended polynucleotide primercontaining a sequence identical to and/or complementary with at leastthat portion of the ligated third or fourth sequences complementary tothe separate portions of the analyte.

Another analytical method in accordance with the present inventioncomprises contacting a sample suspected of containing an analyte withfirst and second polynucleotide probes. The first probe comprises (1) afirst flanking sequence partially or fully complementary to a secondflanking sequence in the second probe and (2) a sequence complementaryto a portion of the analyte other than a portion partially or fullycomplementary to the second probe. The first and second probes areligatable, or capable of being rendered ligatable, to one another onlywhen bound to the analyte. The method further comprises forming in thepresence of nucleoside triphosphates and template dependentpolynucleotide polymerase an extension of a polynucleotide primer atleast a portion of which can be hybridized with the flanking sequence ofthat probe which is ligated to the 3'-end of the other probe. Extendedpolynucleotide primer and/or ligated first and second polynucleotideprobes are then detected.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a dot-blot hybridization of ³² P oligomer 5 to single primerpolynucleotide amplification products.

FIG. 2 is a dot-blot hybridization of a ³² P oligomer 9 to single primerpolynucleotide amplification products.

FIG. 3 is a dot-blot hybridization of ³² P oligomer 5 to single primerpolynucleotide amplification products.

FIG. 4 is a schematic representation of one embodiment of the presentinvention.

FIG. 5 is an example of the determination of a polynucleotide analyte inaccordance with the present invention.

FIG. 6 is an alternate embodiment of the present invention utilizingcertain labels.

FIG. 7 is an alternate embodiment of the present invention.

FIG. 8 is an schematic representation of a method for target dependentformation of hairpin molecule substrate capable of single primeramplification in accordance with the present invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present method allows the production of at least one copy andpreferably multiple copies of a single stranded polynucleotide. Thesingle stranded polynucleotide can be present in a medium or can beformed by the presence of a polynucleotide analyte in a sample suspectedof containing the analyte. In the production of multiple copies of thesingle stranded polynucleotide the following components are provided:(i) the single stranded polynucleotide, (ii) a polynucleotide primer atleast the 3'-end of which has at least a 10 base, preferably at least a15 base sequence hybridizable with a second flanking sequence connectedto the 3'-end of the single stranded polynucleotide, (iii) nucleosidetriphosphates, and (iv) template-dependent polynucleotide polymerase.The second flanking sequence connected to the single strandedpolynucleotide sequence is usually at least 65% complementary with atleast a 10 base, preferably a 15 base, first flanking sequence connectedto the 5'-end of the single stranded polynucleotide. After extension ofthe primer along the single stranded polynucleotide and the firstflanking sequence, the extended polynucleotide sequence is dissociatedfrom the single stranded polynucleotide. Extension and dissociation arerepeated until a desired number of copies is obtained.

One aspect of the invention comprises a determination of apolynucleotide analyte by causing the formation of a single strandedpolynucleotide and initiating the above described method for producingmultiple copies. In this method the single stranded polynucleotide isflanked by first and second polynucleotide flanking sequences thatdiffer from the sequence of the analyte or a sequence complementary tothe analyte. The presence of the analyte is determined by detection asequence located at or beyond the 3' end of the first flanking sequencelinked to a sequence located at or beyond the 5' end of the secondflanking sequence.

Before proceeding further with a description of the specific embodimentsof the present invention, a number of terms will be defined.

Polynucleotide analyte--a compound or composition to be measured whichis a polymeric nucleotide having about 20 to 500,000 or morenucleotides, usually about 100 to 200,000 nucleotides, more frequently500 to 15,000 nucleotides. The polynucleotide analytes include nucleicacids from any source in purified or unpurified form including DNA(dsDNA and ssDNA) and RNA, including t-RNA, m-RNA, r-RNA, mitochondrialDNA and RNA, chloroplast DNA and RNA, DNA-RNA hybrids, or mixturesthereof, genes, chromosomes, plasmids, the genomes of biologicalmaterial such as microorganisms, e.g., bacteria, yeasts, viruses,viroids, molds, fungi, plants, animals, humans, and fragments thereof,and the like. The polynucleotide analyte can be only a minor fraction ofa complex mixture such as a biological sample. The analyte can beobtained from various biological material by procedures well known inthe art. Some examples of such biological material by way ofillustration and not limitation are disclosed in Table I below.

                  TABLE I                                                         ______________________________________                                        Microorganisms of interest include:                                           ______________________________________                                        Corynebacteria                                                                Corynebacterium diphtheria                                                    Pneumococci                                                                   Diplococcus pneumoniae                                                        Streptococci                                                                  Streptococcus pyrogenes                                                       Streptococcus salivarus                                                       Staphylococci                                                                 Staphylococcus aureus                                                         Staphylococcus albus                                                          Neisseria                                                                     Neisseria meningitidis                                                        Neisseria gonorrhea                                                           Enterobacteriaciae                                                            Escherichia coli                                                              Aerobacter aerogenes                                                                            The colliform                                               Klebsiella pneumoniae                                                                           bacteria                                                    Salmonella typhosa                                                            Salmonella choleraesuis                                                                         The Salmonellae                                             Salmonella typhimurium                                                        Shigella dysenteria                                                           Shigella schmitzii                                                            Shigella arabinotarda                                                                           The Shigellae                                               Shigella flexneri                                                             Shigella boydii                                                               Shigella sonnei                                                               Other enteric bacilli                                                         Proteus vulgaris                                                              Proteus mirabilis Proteus species                                             Proteus morgani                                                               Pseudomonas aeruginosa                                                        Alcaligenes faecalis                                                          Vibrio cholerae                                                               Hemophilus-Bordetella group                                                                     Rhizopus oryzae                                             Hemophilus influenza, H. ducryi                                                                 Rhizopus   Phycomycetes                                                       arrhizua                                                    Hemophilus hemophilus                                                                           Rhizopus nigricans                                          Hemophilus aegypticus                                                                           Sporotrichum schenkii                                       Hemophilus parainfluenza                                                                        Flonsecaea pedrosoi                                         Bordetella pertussis                                                                            Fonsecacea compact                                          Pasteurellae      Fonsecacea dermatidis                                       Pasteurella pestis                                                                              Cladosporium carrionii                                      Pasteurella tulareusis                                                                          Phialophora verrucosa                                       Brucellae         Aspergillus nidulans                                        Brucella melitensis                                                                             Madurella mycetomi                                          Brucella abortus  Madurella grisea                                            Brucella suis     Allescheria boydii                                          Aerobic Spore-forming Bacilli                                                                   Phialophora jeanselmei                                      Bacillus anthracis                                                                              Microsporum gypseum                                         Bacillus subtilis Trichophyton mentagrophytes                                 Bacillus megaterium                                                                             Keratinomyces ajelloi                                       Bacillus cereus   Microsporum canis                                           Anaerobic Spore-forming Bacilli                                                                 Trichophyton rubrum                                         Clostridium botulinum                                                                           Microsporum adouini                                         Clostridium tetani                                                                              Viruses                                                     Clostridium perfringens                                                                         Adenoviruses                                                Clostridium novyi Herpes Viruses                                              Clostridium septicum                                                                            Herpes simplex                                              Clostridium histolyticum                                                                        Varicella (Chicken pox)                                     Clostridium tertium                                                                             Herpes Zoster (Shingles)                                    Clostridium bifermentans                                                                        Virus B                                                     Clostridium sporogenes                                                                          Cytomegalovirus                                             Mycobacteria      Pox Viruses                                                 Mycobacterium tuberculosis hominis                                                              Variola (smallpox)                                          Mycobacterium bovis                                                                             Vaccinia                                                    Mycobacterium avium                                                                             Poxvirus bovis                                              Mycobacterium leprae                                                                            Paravaccinia                                                Mycobacterium paratuberculosis                                                                  Molluscum contagiosum                                       Actinomycetes (fungus-like bacteria)                                                            Picornaviruses                                              Actinomyces Isaeli                                                                              Poliovirus                                                  Actinomyces bovis Coxsackievirus                                              Actinomyces naeslundii                                                                          Echoviruses                                                 Nocardia asteroides                                                                             Rhinoviruses                                                Nocardia brasiliensis                                                                           Myxoviruses                                                 The Spirochetes   Influenza (A, B, and C)                                     Treponema pallidum                                                                       Spirillum minus                                                                          Parainfluenza (1-4)                                     Treponema pertenue                                                                       Streptobacilius                                                                          Mumps Virus                                                        monoiliformis                                                                            Newcastle Disease Virus                                 Treponema carateum                                                                              Measles Virus                                               Borrelia recurrentis                                                                            Rinderpest Virus                                            Leptospira icterohemorrhagiae                                                                   Canine Distemper Virus                                      Leptospira canicola                                                                             Respiratory Syncytial Virus                                 Trypanasomes      Rubella Virus                                               Mycoplasmas       Arboviruses                                                 Mycopiasma pneumoniae                                                         Other pathogens   Eastern Equine Eucephalitis Virus                           Listeria monocytogenes                                                                          Western Equine Eucephalitis Virus                           Erysipelothrix rhusiopathiae                                                                    Sindbis Virus                                               Streptobacillus moniliformis                                                                    Chikugunya Virus                                            Donvania granulomatis                                                                           Semliki Forest Virus                                        Bartonella bacilliformis                                                                        Mayora Virus                                                Rickettsiae (bacteria-like parasites)                                                           St. Louis Encephalitis Virus                                Rickettsia prowazekii                                                                           California Encephalitis Virus                               Rickettsia mooseri                                                                              Colorado Tick Fever Virus                                   Rickettsia rickettsii                                                                           Yellow Fever Virus                                          Rickettsia conori Dengue Virus                                                Rickettsia australis                                                                            Reoviruses                                                  Rickettsia sibiricus                                                                            Reovirus Types 1-3                                                            Retroviruses                                                Rickettsia akari  Human Immunodeficiency                                                        Viruses (HIV)                                               Rickettsia tsutsugamushi                                                                        Human T-cell Lymphotrophic                                                    Virus I & II (HTLV)                                         Rickettsia burnetti                                                                             Hepatitis                                                   Rickettsia quintana                                                                             Hepatitis A Virus                                           Chlamydia (unclassifiable parasites                                                             Hepatitis B Virus                                           bacterial/viral)  Hepatitis nonA-nonB Virus                                   Chlamydia agents (naming uncertain)                                                             Tumor Viruses                                               Fungi             Rauscher Leukemia Virus                                     Cryptococcus neoformans                                                                         Gross Virus                                                 Blastomyces dermatidis                                                                          Maloney Leukemia Virus                                      Hisoplasma capsulatum                                                         Coccidioides immitis                                                                            Human Papilloma Virus                                       Paracoccidioides brasiliensis                                                 Candida albicans                                                              Aspergillus fumigatus                                                         Mucor corymbifer                                                              (Absidia corymbifera)                                                         ______________________________________                                    

The polynucleotide analyte, where appropriate, may be treated to cleavethe analyte to obtain a fragment that contains a target polynucleotidesequence, for example, by shearing or by treatment with a restrictionendonuclease or other site specific chemical cleavage method.

For purposes of this invention, the polynucleotide analyte, or thecleaved fragment obtained from the polynucleotide analyte, will usuallybe at least partially denatured or single stranded or treated to renderit denatured or single stranded. Such treatments are well-known in theart and include, for instance, heat or alkali treatment. For example,double stranded DNA can be heated at 90°-100° C. for a period of about 1to 10 minutes to produce denatured material.

Target polynucleotide sequence--a sequence of nucleotides to beidentified, the identity of which is known to an extent sufficient toallow preparation of polynucleotide probes that are complementary to andwill hybridize with at least a portion of such target sequence. Thetarget polynucleotide sequence usually will contain from about 12 to1000 or more nucleotides, preferably 20 to 100 nucleotides. The targetpolynucleotide sequence is frequently a part of the polynucleotideanalyte. The target polynucleotide sequence will generally be a fractionof a larger molecule or it may be substantially the entire molecule. Theminimum number of nucleotides in the target polynucleotide sequence willbe selected to assure that the presence of target polynucleotidesequence in a sample will be a specific indicator of the presence ofpolynucleotide analyte in a sample. Very roughly, the sequence lengthwill usually be greater than about 1.6 log L nucleotides where L is thenumber of base pairs in the genome of the biologic source of the sample.The maximum number of nucleotides in the target sequence will normallybe governed by the length of the polynucleotide analyte and its tendencyto be broken by shearing, by endogenous nucleases or by reagents used tocleave the target sequence.

Single stranded polynucleotide--a natural or synthetic sequence ofnucleotides that exists naturally or is preformed or is formed from aprobe as the result of the presence of an analyte. It will normally becomprised at least of a sequence that is identical to or complementarywith at least a portion of the target polynucleotide sequence and mayalso contain one or more sequences which, when bound to theircomplementary sequences, are specific binding sites for receptors suchas repressors, restriction enzymes, and the like. The single strandedpolynucleotide is flanked by a first flanking sequence and a secondflanking sequence which have at least 50% complementary base sequences,usually at least 65% complementary base sequences, often at least 80%complementary base sequences. The first and second flanking sequencesare attached to the 3'-end and 5'-end, respectively, of the singlestranded polynucleotide and each comprises at least ten, preferably atleast 15, nucleotides, deoxynucleotides, and/or derivatives thereof.

The single stranded polynucleotide together with the flanking sequencesand polynucleotides attached thereto will usually contain from 30 to3,000 nucleotides, preferably 50 to 500 nucleotides. The single strandedpolynucleotide can be RNA or DNA and can also be a syntheticoligonucleotide. When the single stranded polynucleotide flanked by thefirst and second sequences is hybridized with a complementary strand, itwill frequently form inverted repeats.

Polynucleotide primer--a polynucleotide containing a sequence at its3'-end hybridizable with the second flanking sequence at the 3'-end ofthe single stranded polynucleotide. The number of nucleotides in thepolynucleotide primer that are hybridizable with the second flankingsequence should be such that stringency conditions used to hybridize thepolynucleotide primer will prevent excessive random non-specifichybridization. Usually, the number of nucleotides in the polynucleotideprimer will be at least as great as in the second flanking sequence,namely, at least ten nucleotides, preferably at least 15 nucleotides andgenerally from about 10 to 2,000, preferably 20 to 100, nucleotides.

Member of a specific binding pair ("sbp member")--one of two differentmolecules, having an area on the surface or in a cavity whichspecifically binds to and is thereby defined as complementary with aparticular spatial and polar organization of the other molecule. Themembers of the specific binding pair are referred to as ligand andreceptor (antiligand). These may be members of an immunological pairsuch as antigen-antibody, or may be operator-repressor,nuclease-nucleotide biotin-avidin, hormones-hormone receptors, nucleicacid duplexes, IgG-protein A, DNA-DNA, DNA-RNA, and the like.

Ligand--any compound for which a receptor naturally exists or can beprepared.

Receptor ("antiligand")--any compound or composition capable ofrecognizing a particular spatial and polar organization of a molecule,e.g., epitopic or determinant site. Illustrative receptors includenaturally occurring receptors, e.g., thyroxine binding globulin,antibodies, enzymes, Fab fragments, lectins, nucleic acids, repressors,protection enzymes, protein A, complement component Clq, DNA bindingproteins or ligands and the like.

Small organic molecule--a compound of molecular weight less than 1500,preferably 100 to 1000, more preferably 300 to 600 such as biotin,fluorescein, rhodamine and other dyes, tetracycline and other proteinbinding molecules, and haptens, etc. The small organic molecule canprovide a means for attachment of a nucleotide sequence to a label or toa support.

Support or surface--a porous or non-porous water insoluble material. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; glass available as Bioglass, ceramics, metals, andthe like. Natural or synthetic assemblies such as liposomes,phospholipid vesicles, and cells can also be employed.

Binding of sbp members to the support or surface may be accomplished bywell-known techniques, commonly available in the literature. See, forexample, "Immobilized Enzymes," Ichiro Chibata, Halsted Press, New York(1978) and Cuatrecasas, J. Biol. Chem., 245:3059 (1970). The surface canhave any one of a number of shapes, such as strip, rod, particle,including bead, and the like.

The surface will usually be polyfunctional or be capable of beingpolyfunctionalized or be capable of binding an oligonucleotide or an sbpmember through specific or non-specific covalent or non-covalentinteractions. A wide variety of functional groups are available or canbe incorporated. Functional groups include carboxylic acids, aldehydes,amino groups, cyano groups, ethylene groups, hydroxyl groups, mercaptogroups and the like. The manner of linking a wide variety of compoundsto particles is well known and is amply illustrated in the literature.See for example Cuatrecasas, J. Biol. Chem., 245,3059 (1970). The lengthof a linking group to the oligonucleotide or sbp member may vary widely,depending upon the nature of the compound being linked, the effect ofthe distance between the compound being linked and the particle on thehybridization of the sequences and the like. The oligonucleotide or sbpmember will be substantially bound to the outer surface of the particle.

Particles employed as the surface can be fluorescent either directly orby virtue of fluorescent compounds or fluorescers bound to the particlein conventional ways. The fluorescers will usually be dissolved in orbound covalently or non-covalently to the particle and will frequentlybe substantially uniformly bound through the particle. Fluoresceinatedlatex particles are taught in U.S. Pat. No. 3,853,987.

Label or reporter group--A member of the signal producing system that isconjugated to or becomes bound to a probe and is capable of beingdetected directly or, through a specific binding reaction, can produce adetectible signal. Labels include a polynucleotide primer or specificpolynucleotide sequence that can provide a template for amplification orligation or act as a ligand such as for a repressor protein. Preferably,one of the polynucleotide probes will have, or be capable of having, alabel. In general, any label that is detectable can be used. The labelcan be isotopic or nonisotopic, usually non-isotopic, and can be acatalyst, dye, fluorescent molecule, chemiluminescer, coenzyme, enzyme,substrate, radioactive group, a particle such as latex or carbonparticle, metal sol, crystallite, liposome, cell, etc., which may or maynot be further labeled with a dye, catalyst or other detectible group,and the like. The label is a member of a signal producing system and cangenerate a detectable signal either alone or together with other membersof the signal producing system. The label can be bound directly to anucleotide sequence or can become bound thereto by being bound to an sbpmember complementary to an sbp member that is bound to a nucleotidesequence.

Signal Producing System--The signal producing system may have one ormore components, at least one component being the label or reportergroup. The signal producing system generates a signal that relates tothe presence or amount of polynucleotide analyte in a sample. The signalproducing system includes all of the reagents required to produce ameasurable signal. When the label is not conjugated to a nucleotidesequence, the label is normally bound to an sbp member complementary toan sbp member that is bound to or part of a nucleotide sequence. Othercomponents of the signal producing system may be included in a developersolution and can include substrates, enhancers, activators,chemiluminiscent compounds, cofactors, inhibitors, scavengers, metalions, specific binding substances required for binding of signalgenerating substances, and the like. Other components of the signalproducing system may be coenzymes, substances that react with enzymicproducts, other enzymes and catalysts, and the like. The signalproducing system provides a signal detectable by external means, by useof electromagnetic radiation, desirably by visual examination.

The signal-producing system can include at least one catalyst, usuallyan enzyme, and at least one substrate and may include two or morecatalysts and a plurality of substrates, and may include a combinationof enzymes, where the substrate of one enzyme is the product of theother enzyme. The operation of the signal producing system is to producea product which provides a detectable signal related to the amount ofpolynucleotide analyte in the sample.

A large number of enzymes and coenzymes useful in a signal producingsystem are indicated in U.S. Pat. No. 4,275,149, columns 19 to 23, andU.S. Pat. No. 4,318,980, columns 10 to 14, which disclosures areincorporated herein by reference. A number of enzyme combinations areset forth in U.S. Pat. No. 4,275,149, columns 23 to 28, whichcombinations can find use in the subject invention. This disclosure isincorporated herein by reference.

Of particular interest are enzymes which involve the production ofhydrogen peroxide and the use of the hydrogen peroxide to oxidize a dyeprecursor to a dye. Particular combinations include saccharide oxidases,e.g., glucose and galactose oxidase, or heterocyclic oxidases, such asuricase and xanthine oxidase, coupled with an enzyme which employs thehydrogen peroxide to oxidize a dye precursor, that is, a peroxidase suchas horse radish peroxidase, lactoperoxidase, or microperoxidase.Additional enzyme combinations may be found in the subject matterincorporated by reference. When a single enzyme is used as a label,other enzymes may find use such as hydrolases, transferases, andoxidoreductases, preferably hydrolases such as alkaline phosphatase andβ-galactosidase. Alternatively, luciferases may be used such as fireflyluciferase and bacterial luciferase.

Illustrative coenzymes which find use include NAD H!; NADP H!, pyridoxalphosphate; FAD H!; FMN H!, etc., usually coenzymes involving cyclingreactions, see particularly U.S. Pat. No. 4,318,980.

The product of the enzyme reaction will usually be a dye or fluorescer.A large number of illustrative fluorescers are indicated in U.S. Pat.No. 4,275,149, columns 30 and 31, which disclosure is incorporatedherein by reference.

The signal producing system can include one or more particles, which areinsoluble particles of at least about 50 nm and not more than about 50microns, usually at least about 100 nm and less than about 25 microns,preferably from about 0.2 to 5 microns, diameter. The particle may beorganic or inorganic, porous or non-porous, preferably of a densityapproximating water, generally from about 0.7 to about 1.5 g/ml, andcomposed of material that can be transparent, partially transparent, oropaque.

The organic particles will normally be comprised of polymers, eitheraddition or condensation polymers, which are readily dispersible in theassay medium. The surface of particles will be adsorptive orfunctionalizable so as to bind, either directly or indirectly, anoligonucleotide or an sbp member. The nature of particles is describedabove.

Fluorescers of interest will generally emit light at a wavelength above350 nm, usually above 400 nm and preferably above 450 nm. Desirably, thefluorescers have a high quantum efficiency, a large Stokes shift and arechemically stable under the conditions of their conjugation and use. Theterm fluorescer is intended to include substances that emit light uponactivation by electromagnetic radiation or chemical activation andincludes fluorescent and phosphorescent substances, scintillators, andchemiluminescent substances.

Fluorescers of interest fall into a variety of categories having certainprimary functionalities. These primary functionalities include 1- and2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p'-diaminostilbenes immines,anthracenes, oxacarboxyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinal,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene,7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate,strophanthidin, porphyrins, triarylmethanes, flavin and rare earthchelates oxides and salts. Exemplary fluorescers are enumerated in U.S.Pat. No. 4,318,707, columns 7 and 8, the disclosure of which isincorporated herein by reference.

Additionally, energy absorbent or quenching particles can be employedwhich are solid insoluble particles of at least about 50 nm in diametercapable of quenching the fluorescence of the fluorescent particle whenwithin the distance resulting from hybridization of a probe with thepolynucleotide analyte or from specific binding between members ofspecific binding pairs. The quenching particle may be the same ordifferent, usually different, from the fluorescent particle. Normally,the quenching particle will provide a substantial quenching at adistance of more than about 50 Å, preferably more than about 500 Å, morepreferably more than about 2000 ÅA, where the distance is measured fromthe surfaces of the particles.

Many different types of particles may be employed for modulating lightemission. Of particular interest are carbon particles, such as charcoal,lamp black, graphite, colloidal carbon and the like. Besides carbonparticles metal sols may also find use, particularly of the noblemetals, gold, silver, and platinum. Other metal-derived particles mayinclude metal sulfides, such as lead, silver or copper sulfides or metaloxides, such as iron or copper oxide.

An alternative source of light as a detectible signal is achemiluminescent source. The chemiluminescent source involves a compoundwhich becomes electronically excited by a chemical reaction and may thenemit light which serves as the detectible signal or donates energy to afluorescent acceptor.

A diverse number of families of compounds have been found to providechemiluminescence under a variety of conditions. One family of compoundsis 2,3-dihydro-1,4-phthalazinedione. The most popular compound isluminol, which is the 5-amino analog of the above compound. Othermembers of the family include the 5-amino-6,7,8-trimethoxy- and thedimethylamine- ca!benz analog. These compounds can be made to luminescewith alkaline hydrogen peroxide or calcium hypochlorite and base.Another family of compounds is the 2,4,5-triphenylimidazoles, withlophine as the common name for the parent product. Chemiluminescentanalogs include para-dimethylamino- and para-methoxy-substituents.Chemiluminescence may also be obtained with oxilates, usually oxalyl,active esters, e.g., p-nitrophenyl and a peroxide, e.g., hydrogenperoxide, under basic conditions. Alternatively, luciferins may be usedin conjunction with luciferase or lucigenins.

Ancillary Materials--Various ancillary materials will frequently beemployed in the assay in accordance with the present invention. Forexample, buffers will normally be present in the assay medium, as wellas stabilizers for the assay medium and the assay components.Frequently, in addition to these additives, proteins may be included,such as albumins, organic solvents such as formamide, quaternaryammonium salts, polycations such as dextran sulfate, surfactants,particularly non-ionic surfactants, binding enhancers, e.g.,polyalkylene glycols, or the like.

Nucleoside triphosphates--a nucleoside having a 5' triphosphatesubstituent, preferably a deoxynucleoside triphosphate. The nucleosidesare pentose sugar derivatives of nitrogenous bases of either purine orpyrimidine derivation, covalently bonded to the 1'-carbon of the pentosesugar. The purine bases include adenine(A), guanine(G), inosine, andderivatives and analogs thereof. The pyrimidine bases include cytosine(C), thymine (T), uracil (U), and derivatives and analogs thereof.

The derivatives and analogs are exemplified by those that are recognizedand polymerized in a similar manner to the underivitized nucleosidetriphosphates. Examples of such derivatives or analogs by way ofillustration and not limitation are those which are modified with areporter group, biotinylated, amine modified, radiolabeled, alkylated,and the like and also include thiophosphate, phosphite, ring atommodified derivatives, and the like. The reporter group can be afluorescent group such as fluoroscein, a chemiluminescent group such asluminol, a terbium chelator such as N-(hydroxyethyl)ethylenediaminetriacetic acid that is capable of detection by delayedfluorescence, and the like.

Polynucleotide polymerase--a catalyst, usually an enzyme, for forming anextension of the polynucleotide primer along a DNA or RNA templateincluding the single stranded polynucleotide where the extension iscomplementary thereto. The polynucleotide polymerase is a templatedependent polynucleotide polymerase and utilizes the nucleosidetriphosphates as building blocks for extending the 3'-end of thepolynucleotide primer to provide a sequence complementary with thesingle stranded polynucleotide. Usually, the catalysts are enzymes, suchas RNA polymerases, preferably DNA polymerases such as, for example,prokaryotic DNA polymerase (I, II, or III), T4 DNA polymerase, T7 DNApolymerase, Klenow fragment, reverse transcriptase, RNA replicases, andthe like derived from any source such as cells, bacteria, such as E.coli, plants, animals, virus, thermophilic bacteria, and so forth.

Wholly or partially sequentially--when the sample and various agentsutilized in the present invention are combined other than concomitantly(simultaneously), one or more may be combined with one or more of theremaining agents to form a subcombination. Each subcombination can thenbe subjected to one or more steps of the present method. Thus, each ofthe subcombinations can be incubated under conditions to achieve one ormore of the desired results.

Hybridization (hybridizing) and binding--in the context of nucleotidesequences these terms are used interchangeably herein. The ability oftwo nucleotide sequences to hybridize with each other is based on thedegree of complementarity of the two nucleotide sequences, which in turnis based on the fraction of matched complementary nucleotide pairs. Themore nucleotides in a given sequence that are complementary to anothersequence, the greater the degree of hybridization of one to the other.The degree of hybridization also depends on the viscosity conditions orstringency which include temperature, solvent ratios, saltconcentrations, and the like.

First polynucleotide probe--a compound having a polynucleotide sequenceat its 3'-end (3'-target binding sequence) at least a portion of suchsequence, preferably all of such sequence, capable of hybridizing with aportion of the target polynucleotide analyte by virtue of beingpartially or completely, usually completely, complementary to a regionof the target polynucleotide analyte such that the first polynucleotideprobe will become bound to such region of the target polynucleotideanalyte. The first polynucleotide probe also comprises a first flankingsequence that is at least 50% complementary, usually at least 65%complementary, to a second flanking sequence of a second polynucleotideprobe. The first polynucleotide probe may contain additional sequenceslocated between the target polynucleotide binding sequence and the firstflanking sequence and attached to the first flanking sequence distal tothe target polynucleotide binding sequence.

The major criteria for choosing the first polynucleotide probe are: (1)The 3'-end target binding sequence should be reliable, that is, itshould be closely or exactly complementary with at least a portion ofthe target nucleotide sequence and should be of sufficient length toprovide stable and specific binding. (2) The 3'-end must form, or becapable of forming, a free 3'-hydroxyl group. The minimum first flankingsequence will usually be at least 10, preferably at least 15,nucleotides in length. Additional sequences, usually located between the3'-end target binding sequence and the first flanking sequence areselected to increase the distance between the two groups and thusprovide for greater DNA synthesis during amplification and to providefor receptor binding sites to permit detection of the amplified product.In general, the first polynucleotide probe will be about 30 to 5,000nucleotides, more frequently 40 to 1,000 nucleotides in length. Thecombined length of the hybridizing portion of the first and secondpolynucleotide probes is at least about 20 nucleotides, preferably about40 to 2,000 nucleotides, in length. With biologically synthesizedpolynucleotides random fragments of unknown sequences may be usedprovided, however, that nucleic acids are single stranded andcomplementary to the target nucleotide sequences or the polynucleotideanalyte.

Second polynucleotide probe--The second polynucleotide probe has asequence at its 5'-end, at least a portion and preferably all of whichis capable of hybridizing with the target polynucleotide analyte at aregion other than that with which the first polynucleotide probehybridizes (5'-target binding sequence). The second polynucleotide probehas a sequence that is at least 50% complementary, usually at least 65%complementary to the first flanking sequence of the first polynucleotideprobe and is designated as the second flanking sequence. Thus, the firstand second polynucleotide probes each have a polynucleotide sequencethat is at least partially complementary to a sequence in the other. Thesecond polynucleotide probe may contain additional receptor binding orspacer sequences located between the target polynucleotide bindingsequence and the second flanking sequence and attached to the secondflanking sequence distal to the target polynucleotide binding sequence.

The two regions of the target polynucleotide analyte complementary tothe first and second polynucleotide probes will normally be inreasonable proximity to one another to ensure that a substantialfraction of the analyte will have the two regions linked. The tworegions may be within 0 to 50 nucleotides, preferably 0 to 1nucleotides. Where the regions are separated by more than onenucleotide, it will frequently be desirable to extend the first probewhen bound to the target by means of a nucelotide polymerase andnucleoside triphosphates and thereby reduce the distance between theprobes.

Non-contiguous--the probes are hybridized to non-contiguous portions ofthe target nucleotide sequence, there being at least one nucleotidepresent in the target polynucleotide sequence between the hybridized 5'terminus of the first polynucleotide probe and the 3'-end of the secondpolynucleotide probe.

Contiguous--the probes are considered to be contiguous when there are nonucleotides between the 5'-end of the first probe sequence and the3'-end of the second probe sequence, when these nucleotide sequences arehybridized with the target polynucleotide analyte.

Copy--means a sequence that is a direct copy of a single strandedpolynucleotide as differentiated from a sequence that is complementaryto the sequence of such single stranded polynucleotide. In the presentinvention a complementary sequence of a single stranded polynucleotideis produced initially as the result of the extension of thepolynucleotide primer and a sequence that is a direct copy of the singlestranded polynucleotide is subsequently obtained from the aforementionedcomplementary sequence.

Covalently attaching--forming a chemical bond between the firstpolynucleotide probe and the second polynucleotide probe.

The chemical bond can be formed whether the probes are contiguous or notwhen the probes are bound to the target polynucleotide analyte, usuallywhen separated by 0 or 1 nucleotide. Covalent attachment can be achievedenzymatically, for example by utilizing a ligase. Prior to ligating theprobes, the probe having a 3' terminus extendable in the direction ofthe other probe hybridized with the target polynucleotide analyte may betreated to render it contiguous or effectively contiguous with the otherprobe. The probes are effectively contiguous, for example, whenenzymatic ligation or a chemical coupling can take place. Generally, theprobes are effectively contiguous when they are within 1 to 3nucleotides apart. Chain extension may be achieved, for example, byadding a polynucleotide polymerase and nucleoside triphosphates or byligating to one of the probes a nucleotide sequence complementary to thenon-contiguous region of the target polynucleotide analyte betweenhybridized probes. The covalent attachment may be achieved chemically byforming chemical bonds between the phosphate moieties of the probes.

Means for extending a probe--in order to ligate the probes when theprobes are bound with the target polynucleotide sequence it is oftendesirable to render the probes contiguous. As explained above, the probehaving a 3'-terminus extendable in the direction of the other probe canbe extended by combining the probe hybridized to the targetpolynucleotide analyte with a polynucleotide polymerase and nucleosidetriphosphates under conditions for extending the probe. Alternatively, anucleotide sequence complementary to the non-contiguous portion of thetarget polynucleotide analyte between the probes can be ligated to oneof the probes.

One embodiment of the present invention concerns a method of producingat least one copy of a single stranded polynucleotide or of a sequencecomplementary thereto. The method comprises forming in the presence ofnucleoside triphosphates and template dependent polynucleotidepolymerase an extension of a polynucleotide primer having a sequencehybridizable with a first flanking sequence at the 3'-end of the singlestranded polynucleotide, wherein the first flanking sequence is at leastpartially complementary with a second flanking sequence of the singlestranded polynucleotide and dissociating the extended polynucleotideprimer and the single stranded polynucleotide. The above steps arerepeated to yield the appropriate copy.

Generally, a combination is provided in a liquid medium which comprises(1) a single stranded polynucleotide having a polynucleotide sequenceflanked at each end by internally hybridizable first and second flankingsequences, (2) a polynucleotide primer at least a 10 base portion ofwhich at its 3'-end is complementary to that member of the first andsecond flanking sequences that is at the 3'-end of the single strandedpolynucleotide, (3) nucleoside triphosphates, and (4) template dependentpolynucleotide polymerase. The combination is incubated under conditionsfor (1) dissociating any internally hybridized first and second flankingsequences, (2) hybridizing the polynucleotide primer with itscomplementary sequence, flanking the single stranded polynucleotide, (3)extending the polynucleotide primer along the single strandedpolynucleotide to provide a first extended polynucleotide primer, (4)dissociating the first extended polynucleotide primer and the singlestranded polynucleotide, (5) hybridizing the first extendedpolynucleotide primer with the polynucleotide primer, (6) extending thepolynucleotide primer along the first extended polynucleotide primer toprovide a second extended polynucleotide primer, (7) dissociating thesecond extended polynucleotide primer from the first extendedpolynucleotide primer, and (8) repeating steps (5)-(7) above.

An embodiment of the present invention is illustrated in FIG. 4 by wayof example and not limitation.

Molecule I can be, for example, double stranded DNA having an invertedrepeat with complementary sequences 1a and 1b. The DNA can be renderedsingle stranded to yield molecule II, or molecule II can be RNA havingsequences 1a and 1b. Hybridization of polynucleotide primer 1c withmolecule II yields molecule III. Primer 1c has substantially the same ora similar polynucleotide sequence as sequence 1a. In the presence of DNApolymerase and nucleoside triphosphates primer 1c is extended alongmolecule II to yield molecule IV. Dissociation of molecule IV yieldssingle stranded IVa and IVb. Molecule IVa is the unchanged molecule IIand has complementary sequences 1a and 1b and molecule IVb hascomplementary sequences 1c and 1d. As is evident 1ccorresponds to 1a,and 1d corresponds to 1b. Polynucleotide primer 1c can be hybridized toregion 1b of IVa and to region 1d of IVb to yield molecules Va (III) andVb, respectively. Extension of primer 1c along Va and Vb underconditions described above yields molecules VIa (IV) and VIb,respectively. Molecules VIa and VIb can be dissociated to singlestranded polynucleotides, which can then hybridize with primer 1c andthe chain extension can be repeated. In this way multiple copies of theinitial single stranded polynucleotide encompassing the sequence betweenthe sequences 1a and 1b of molecule II, and a sequence complementarythereto, can be obtained.

In carrying out the method an aqueous medium will be employed. Otherpolar cosolvents may also be employed, usually oxygenated organicsolvents of from 1-6, more usually from 1-4, carbon atoms, includingalcohols, ethers and the like. Usually these cosolvents will be presentin less than about 70 weight percent, more usually in less than about 30weight percent.

The pH for the medium will usually be in the range of about 4.5 to 9.5,more usually in the range of about 5.5-8.5, and preferably in the rangeof about 6-8. The pH and temperature are chosen and varied, as the casemay be, so as to provide for either simultaneous or sequentialdissociation of any internally hybridized sequences in the singlestranded polynucleotide sequence, hybridization of the polynucleotideprimer with the single stranded polynucleotide, extension of the primer,dissociation of the extended primer, hybridization of extended primerwith primer, extension of the so-hybridized primer, and dissociation ofextended primer. In some instances, a compromise will be made betweenthese considerations depending on whether the above steps are performedsequentially or simultaneously. Various buffers may be used to achievethe desired pH and maintain the pH during the determination.Illustrative buffers include borate, phosphate, carbonate, Tris,barbital and the like. The particular buffer employed is not critical tothis invention but in individual methods one buffer may be preferredover another.

Moderate temperatures are normally employed for carrying out the method.Desirably constant temperatures during the period for conducting themethod will be used but frequently the medium will be cycled between twoor three temperatures. When constant, the temperature will be near themelting temperature of the complex of the single stranded polynucleotideand the extended polynucleotide primer. The temperatures for the methodwill generally range from about 10° to 100° C., more usually from about20° to 95° C., preferably 35° to 70° C. However, the temperature can bevaried depending on whether the above steps are carried out sequentiallyor simultaneously. For example, relatively low temperatures of fromabout 20° to 40° C. can be employed for the extension steps, whiledenaturation and hybridization can be carried out at a temperature offrom about 50° to 95° C.

The time period for carrying out the method of the invention willgenerally be long enough to achieve a desired number of copies of thesingle stranded polynucleotide or a sequence complementary thereto.This, in turn, depends on the purpose for which the amplification isconducted, such as, for example, an assay for a polynucleotide analyte.Generally, the time period for conducting the method will be from about1 to 10 minutes per cycle and any number of cycles can be used from 1 toas high as 200 or more, usually 1 to 80, frequently 20-80. As a matterof convenience it will usually be desirable to minimize the time periodand the number of cycles. In general, the time period for a given degreeof amplification can be shortened, for example, by selectingconcentrations of nucleoside triphosphates sufficient to saturate thepolynucleotide polymerase and by increasing the concentrations ofpolynucleotide polymerase and polynucleotide primer.

The amount of the single stranded polynucleotide which is to be copiedcan be as low as one or two molecules in a sample but will generallyvary from about 10² to 10¹⁰, more usually from about 10³ to 10⁸molecules in a sample. The amount of the polynucleotide primer will beat least as great as the number of copies desired and will usually be10⁻¹⁵ to 10⁻⁹ moles per sample, where the sample is 10-1,000 μL.Usually, the primer will be present in at least 10⁻¹² M, preferably10⁻¹⁰ M, and more preferably at least about 10⁻⁸ M. Preferably, theconcentration of the polynucleotide primer is substantially in excessover, preferably at least 100 times greater than, the concentration ofthe single stranded polynucleotide.

The final concentration of each of the reagents will normally bedetermined empirically to optimize the number of the copies of thetarget sequence.

The concentration of the deoxynucleoside triphosphates in the medium canvary widely; preferably, these reagents are present in an excess amount.The deoxynucleoside triphosphates will usually be present in 10⁻⁶ to10⁻² M, preferably 10⁻⁵ to 10⁻³ M.

The concentration of the template-dependent polynucleotide polymerasewill usually be determined empirically. Preferably, a concentration willbe used that is sufficient such that further increase in theconcentration will not decrease the time for the amplification by over5-fold, preferably 2-fold. The primary limiting factor generally is thecost of the reagent.

The order of combining of the various reagents to form the combinationmay vary. Generally, a single stranded polynucleotide with first andsecond flanking sequences is itself the target polynucleotide sequencein the sample or is formed as a function of the presence of apolynucleotide analyte in the sample. This may be combined with apre-prepared combination of polynucleotide primer, nucleosidetriphosphates, and template-dependent polynucleotide polymerase.However, simultaneous addition of the above, as well as other step-wiseor sequential orders of addition, may be employed.

The concentration and order of addition of reagents and conditions forthe method are governed generally by the desire to maximize the numberof copies of the single stranded polynucleotide sequence and the rate atwhich such copies are formed. Generally, it is desirable to increase thenumber of copies of the single stranded polynucleotide sequence by atleast a factor of 10², preferably a factor of 10⁴, more preferably 10⁶or more.

The present invention has particular application to the determination ordetection of a polynucleotide analyte in a sample. In general, themethod comprises forming as a result of the presence of an analyte asingle stranded polynucleotide flanked by first and secondpolynucleotide flanking sequences. Multiple copies of the singlestranded polynucleotide are then made. Directly or indirectly detectingthe thus formed single stranded polynucleotide indicates the presence ofthe analyte.

The single stranded polynucleotide can be formed by contacting thesample with first and second polynucleotide probes. The first probecomprises a sequence at its 3'-end complementary to a first portion ofone strand of the analyte and a first flanking sequence. The said secondprobe comprises a sequence at its 5'end complementary to a secondportion of the same strand of the analyte and a second flankingsequence. The first and second flanking sequences are at least 50-65%complementary with each other and will frequently be 80-100%complementary. The contact is carried out under conditions for bindingof the first and second probes with the analyte. Then, conditions areprovided for ligating the first and second polynucleotide probes to oneanother only when bound to said analyte.

The order of combining of the various reagents to form the combinationmay vary and can be concomitant or simultaneous or wholly or partiallysequential. Generally, a sample containing an analyte sequence isobtained. This may be combined with a pre-prepared combination of firstand second polynucleotide probes, nucleoside triphosphates, andpolynucleotide polymerase, followed by addition of a ligase. However,simultaneous addition of the above, as well as other step-wise orsequential orders of addition, may be employed. The concentration andorder of addition of reagents and conditions for the method are governedgenerally by the desire to optimize hybridization of all the targetpolynucleotide sequences with the first and second polynucleotide probesand covalent attachment of the so-hybridized first and secondpolynucleotide probes.

One means for covalently attaching the first and second probes whenthese probes are hybridized with the target polynucleotide sequenceinvolves the chain extension of the first probe to render the first andsecond polynucleotide probes contiguous. One means for extending thefirst probe comprises adding a polynucleotide polymerase anddeoxynucleoside triphosphates to the liquid medium and incubating themedium under conditions for forming a chain extension at the 3'-end ofthe first probe to render it contiguous with the second polynucleotideprobe when these probes are hybridized with the analyte.

When the first and second polynucleotide probes are rendered contiguouswhen hybridized with the analyte sequence, the first and second probesare then covalently attached. One method of achieving covalentattachment of the first and second polynucleotide probes is to employenzymatic means. Preferably the medium containing the first and secondpolynucleotide probes hybridized with the analyte sequence can betreated with a ligase, which catalyzes the formation of a phosphodiesterbond between the 5' end of one probe and the 3' end of the other.

Any enzyme capable of catalyzing the reaction of the polynucleotide3'-hydroxyl group can be employed. Examples, by way of illustration andnot limitation, of such enzymes are polynucleotide ligases from anysource such as E coli bacterial ligase, T4 phage DNA ligase, mammalianDNA ligase, and the like. The pH, temperature, solvent, and timeconsiderations will be similar to those described above for the methodof the invention.

Another means for achieving the covalent attachment of the first andsecond polynucleotide probes when the probes are hybridized tonon-contiguous portions of the analyte sequence involves the use of anucleotide sequence that is sufficiently complementary to thenon-contiguous portion of the analyte sequence lying between the firstand second nucleotide probes. For purposes of this description such anucleotide sequence will be referred to as an intervening linkersequence. The linker sequence can be prepared by known methods such asthose described above for the preparation, for example, of the first andsecond polynucleotide probes. The linker sequence can be hybridized tothe analyte sequence between the first and second polynucleotide probes.The linker sequence can then be ligated to both the first and secondpolynucleotide probes utilizing enzymatic means as referred to above. Itis also possible to utilize combinations of linker sequences andpolymerase to achieve a contiguous relationship between the first andsecond polynucleotide probes when these sequences are bound to theanalyte.

Following ligation of the first and second polynucleotide probes whenthese probes are hybridized with the polynucleotide analyte, thehybridized polynucleotides are dissociated. Because each of the probescontains a flanking sequence that is potentially hybridizable with aflanking sequence in the other, the single stranded polynucleotide alsocontains these sequences. Multiple copies of the single strandedpolynucleotide resulting from the ligated probes are then prepared. Inone approach multiple copies of the single stranded polynucleotide areobtained by the procedures described above using a single polynucleotideprimer. In another approach multiple copies of the single strandedpolynucleotide are obtained by using the double primer techniquedescribed in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosures ofwhich are incorporated herein by reference. In still another approachamplification can be achieved as described in U.S. patent applicationSer. No. 076,807 filed Jul. 23, 1987 now U.S. Pat. No. 4,994,368, thedisclosure of which is incorporated herein reference. It will beappreciated by those skilled in the art that other methods of formingmultiple copies can be used in the present invention for detection of ananalyte.

Detection of the first flanking sequence linked to the second flankingsequence or the detection of the single stranded polynucleotide or itscomplementary sequence indicates the presence of the polynucleotideanalyte in the sample.

An example of the determination of a polynucleotide analyte isillustrated, by way of example and not limitation, in FIG. 2.

The polynucleotide analyte is represented in VII wherein twopolynucleotide probes are hybridized to a portion of the target analyte.The probes respectively contain sequences 2a and 2b that are at leastpartially complementary to one another. Molecule VII is treated torender the probes ligatable and to ligate the probes. This can beaccomplished as described above and is depicted as molecule VIII in FIG.5. Dissociation of VIII yields molecule IX, which is a single strandedpolynucleotide having two at least partially complementary sequences, 2aand 2b. As can be seen, molecule IX is similar to molecule II in FIG. 4and can be combined with a polynucleotide primer 2c, which hybridizeswith sequence 2b of molecule IX to give molecule X. Chain extension ofprimer 2c as described above yields molecule XI. Dissociation of XIfollowed by hybridization with primer 2c and chain extension yieldsmultiple copies of molecule IX or a sequence complementary thereto. Thesingle stranded polynucleotide is detected either as a single strand orhybridized with its complementary strand. The presence of ligated probesindicates the presence of the polynucleotide analyte in the sample.

In carrying out the method of the invention as applied to the detectionof a polynucleotide analyte, an aqueous medium is employed, which mayfurther contain other polar solvents as described above. The pH,temperature, time, and concentration of reagents generally will be thosedescribed above for the formation of multiple copies of a singlestranded polynucleotide.

The pH for the medium will usually be in the range of about 4.5 to 9.5,more usually in the range of about 5.5-8.5, and preferably in the rangeof about 6-8.

The temperatures for the method will generally range from about 20° to90° C., more usually from about 30° to 70° C. preferably 37° to 50° C.

Generally, the time period for conducting the method will be from about5 to 200 min. As a matter of convenience, it will usually be desirableto minimize the time period.

The concentration of the target polynucleotide analyte can be as low aspossibly one molecule, preferably at least 10⁻²¹ M in a sample but willgenerally vary from about 10⁻¹⁴ M to 10⁻¹⁹ M, more usually from about10⁻¹⁶ to 10⁻¹⁹ M. The concentration of the first and secondpolynucleotide probes and the deoxynucleoside triphosphates in themedium can vary widely. Preferably, these reagents will be present inlarge molar excess over the amount of target analyte expected. Thedeoxynucleoside triphosphates will usually be present in 10⁻⁶ to 10⁻² M,preferably 10⁻⁵ to 10⁻³ M. The second polynucleotide probe, as well asthe first polynucleotide probe, will usually be present in at least10⁻¹² M, preferably 10⁻¹⁰ M, more preferably at least about 10⁻⁸ M.

The concentration of the polymerase and any cofactors in the medium canalso vary substantially. These reagents may be present in as low as10⁻¹² M but may be present in a concentration at least as high or higherthan the concentration of the first and second nucleotide probes.

While the concentrations of the various reagents will generally bedetermined by the concentration range of interest of the polynucleotideanalyte, the final concentration of each of the reagents will normallybe determined empirically to optimize the sensitivity of the assay overthe range of interest. The concentration of the other reagents in anassay generally will be determined following the same principles as setforth above for the amplification method. The primary consideration isthat a sufficient number of copies of a single stranded polynucleotidebe produced in relation to the polynucleotide analyte sequence so thatsuch copies can be readily detected and provide an accuratedetermination of the polynucleotide analyte.

After the medium is incubated either concomitantly or sequentially underthe above conditions any single stranded polynucleotide or ligatedtarget binding sequences of the first and second probes present aredetected. The presence of the ligated sequences indicates the presenceof the polynucleotide analyte in the sample. The ligated sequences canbe detected in numerous ways.

In the present method, some of the molecules of the polynucleotideprimer can be labeled with a ligand (B) and other of the molecules ofthe polynucleotide primer can be labeled with a detectable (F) label.The ligand can be a small organic molecule, a polynucleotide sequence, aprotein, or the like. Upon amplification, a mixture of duplexes isobtained (See FIG. 6), some having ligand at both ends, some havingdetectable label at both ends, and some having ligand at one end anddetectable label at the other. The ratios of the products can bemodified by varying the ratio of the two differently labeled primers.The duplexes can be detected by causing the molecule to bind to asurface to which is bound a receptor for the ligand. Duplexes containingthe two primer labels that are shorter than the single strandedpolynucleotide with its first and second flanking sequences can beprevented from binding by using conditions that are stringent enough todissociate only these shorter duplexes. After removal of unboundmaterial, the support is examined for the presence of a detectablelabel. The presence thereof indicating the presence of polynucleotideanalyte in the sample.

In another approach, the internally hybridizable sequences can beselected because a synthetic or natural receptor exists that can bind tothe hybridized sequences. The sequences will usually be introduced byincluding them between the target polynucleotide binding sequence and aflanking sequence of a polynucleotide probe or the single strandedpolynucleotide. Alternatively, they can be introduced as labels at the5'-end of a portion of the polynucleotide primer molecules. Thetetracycline repressor is such a receptor. This protein binds to thetetracycline operator and the hybridized sequences can be selected tocomprise some or all of this operator. The repressor is bound to a solidsupport and used to absorb and concentrate the amplification productfrom the amplification reaction solution. The bound product can then bedetected by staining with a dye such as acridinium orange, by changes ina physical property of the adsorbent such as electrical properties,optical properties, acoustic wave modulation, and the like, or bydetecting the presence of a label bound to another portion of thepolynucleotide primer molecules.

Other operator-repressor pairs can be used including, for example, thelac repressor and operator which have been used as a ligand and receptorfor capture of DNA duplexes and the tryptophane repressor and operator.

In another approach bromodeoxyuridine can be incorporated into a portionof the polynucleotide primer molecules and antibodies tobromodeoxyuridine can be employed. Detection of the bound sequence canbe accomplished by any of the above methods.

In a preferable approach for detection of the single strandedpolynucleotide, the polynucleotides are simultaneously or sequentiallydenatured by heating or use of denaturing solvents and solutes andcaused to bind to a support by, for example, one of the above methods.The support is then contacted with a probe comprised of a nucleic acidsequence and a label or receptor binding site. The nucleic acid sequenceis complementary to at least the portion of the single strandedpolynucleotide that was ligated, or its complementary sequence. Thepresence of the single stranded polynucleotide is then indicated by thepresence of the label or receptor binding site on the support.

In another assay format (as depicted in FIG. 7), the single strandpolynucleotide containing the internally hybridizable sequences 3a and3b can be used as a label. In this method, any means, such as formationof a DNA sandwich, can be used to cause a labeled nucleic acid strand tobind to a surface. In the sandwich method, two probes are used that canbind to a target analyte. One of these probes is bound or can be causedto bind to a solid surface, for example, by the use of legand-receptorbinding, such as biotin-avidin. The other probe carries the singlestranded polynucleotide as a label. After (1) hybridization of theprobes with the target polynucleotide sequence, (2) binding of thecomplex to the surface when one of the probes is not already bound, and(3) washing of the surface, the single stranded polynucleotide is causedto initiate the present amplification process and the products aredetected by any convenient method of detecting specific polynucleotidesequences, including the above described methods for detecting thesingle stranded polynucleotide.

Any standard method for specifically detecting double strand nucleicacid sequences can be used.

One method for detecting nucleic acids is to employ nucleic acid probes.This method generally involves immobilization of the target nucleic acidon a solid support such as nitrocellulose paper, cellulose paper,diazotized paper, or a nylon membrane. After the target nucleic acid isfixed on the support, the support is contacted with a suitably labelledprobe nucleic acid for about ten minutes to forty-eight hours. After theabove time period, the solid support is washed several times to removeunbound probe and the hybridized material is detected by autoradiographyor spectroscopic methods.

One method utilizing probes is described in U.S. patent application Ser.No. 773,386, filed Sep. 6, 1985 now U.S. Pat. No. 4,860,104, thedisclosure of which is incorporated herein by reference. The methodcomprises combining in an assay medium the sample and first and secondpolynucleotide reagents complementary to the nucleic acid fragment. Eachof the first and second reagents hybridize with a different region ofnucleic acid fragment. The first reagent contains means for renderingthe first reagent non-covalently polymerizable. The second reagentcontains means for rendering the second reagent detectable. The sampleand the first and second reagents are combined in the assay medium underconditions for polymerizing the first reagent wherein the second reagentbecomes bound to the polymerized first reagent only when the DNAfragment is present in the sample. A determination is then made as towhether the second reagent has become bound to the polymerized firstreagent.

In order to separate the single stranded polynucleotide from othercomponents in an assay mixture containing a sample it can be desirable,and indeed preferable in some circumstances, that the first or secondpolynucleotide probe or the single stranded polynucleotide has, or iscapable of having, means for immobilizing the sequence. Generally, thismeans for immobilizing involves a support. The sequence in question canbe treated to bind the sequence to a support prior to the use of thissequence in the method of the present invention. Numerous methods areknown for binding nucleotide sequences to solid supports. For examplesee T. Goldkorn et al., Nucleic Acids Research (1986 ) 14:9171-9191 andthe references contained therein. Generally, the procedures forattaching the nucleotide sequence to supports involve chemicalmodifications of some of the nucleotides in the sequence whereby thesequence can then be attached to the support. Preferably, the bondbetween the support and the nucleotide sequence will be covalent, morepreferably involving a linking group between the nucleotide sequence thesupport. For example, the support can be treated to introduce maleimidegroups and the nucleotide sequence can be treated to introduce a thiolgroup. The thiol group is reactive with the activated olefin of themaleimide group and in such a fashion the nucleotide sequence can becovalently bound to the support. Examples of other such linking groupsare cellulose derivatized with diazobenzyloxymethyl groups as describedby Noyes, B. E. and Start, G. R., Cell 5, 301 (1975) and Alwine, J. C.,et al., Proc. Natl. Acad. Sci., U.S.A. 74, 5350 (1977), and cellulosederivatized with o-aminophenylthioether, such as described by Seed, B.,Nucleic Acids Res., 10, 1799 (1982).

If the nucleotide sequence is not initially bound to a support, it maybe desirable that one of the two sequences become bound to a support atsome time during the method of the invention, preferably, prior to thedetermination of whether the first and second target polynucleotidebinding sequences have become covalently attached as a result of thepresence of the polynucleotide analyte. Accordingly, the support and oneof the nucleotide sequences must contain reactive groups which canprovide a linkage between the support and the nucleotide sequence. Thenature of the reactive groups will be such as to be compatible with themethod of the present invention.

One such system is that described above where the support would containmaleimide groups and the nucleotide sequence would contain a thiolgroup. In another embodiment the nucleotide sequence and the support cancontain complementary specific binding pair members such asbiotin-avidin and the like. Thus, the method of the present inventioncan be run in solution and at the appropriate time the support can beintroduced whereupon the complementary sbp members will bind. After thesupport is washed, to remove unbound material, further reactions ordeterminations can be carried out.

Other examples of such systems are repressor-operator interactions whereone of the nucleotide sequences is captured at the solid surface by itssequence specific interaction with a specific repressor or modulatorprotein immobilized on the solid surface. An advantage of thisembodiment of the capture phase is that in some cases release of theoperator DNA from the repressor can be accomplished by treating thecomplex with an inducer molecule. For example, the tetracyclinerepressor may be immobilized on a solid surface so that an operatorsequence present on one or the other of the nucleotide sequences isspecifically captured and retained when the solution is contacted to thesurface. The surface may then be washed to eliminate any non-specificbinding and finally the operator containing nucleotide may be releasedfrom the surface by contacting the repressor-operator complex bound atthe surface with an inducer molecule (tetracycline or one of its activeanalogs in this case).

The inducer molecule may be the "natural inducer" in the sense that itis structurally identical with the molecule in nature that causesdissociation of the biological/regulatory repressor-operator complex orit may be a synthetic analog of the natural inducer with similar orenhanced binding and complex dissociation activity. Examples of theabove include the tetracycline repressor-operator interaction and itsdissociation by tetracycline such as described by Hillen, W., et al., J.Mol. Biol., 169, 707-721 (1983) and Klock, G., et al., J. Bact., 161,326-332 (1985).

In the situation where the nucleotide sequence is covalently attached tothe support, it may be desirable to remove the attached sequence fromthe support, such as, for example, in order to amplify or clone thesequence. In this situation it is desirable to introduce a cleavablegroup between the nucleotide sequence and the support. Exemplary of suchcleavable groups are pyrophosphate linkages, disulfide linkages andrestriction enzyme cleavage sites.

After provision has been made for covalently attaching the first andsecond polynucleotide probes when these sequences are hybridized withthe polynucleotide analyte to produce a single stranded polynucleotideand after amplification of the single stranded polynucleotide ifpresent, the presence of covalently attached first and second targetpolynucleotide binding sequences is determined. The presence of covalentattachment indicates the presence of the polynucleotide analyte in thesample. As mentioned above this determination may involve a nucleotidesequence bound, or capable of becoming bound, to a support. The supportis removed from the medium, washed free of unbound material, and thenexamined for the coupled first and second target polynucleotide bindingsequences, for example, by detecting the presence of a label or areporter group. Generally, this examination involves contacting thesupport with the remaining members of a signal producing system in orderto produce a signal in relation to the presence of the target nucleotidesequence in the sample.

Use of the method in accordance with the present invention allows asupport to be washed under conditions that would normally be morevigorous than those used when hybridization is carried out withoutcovalent attachment. Frequently, the washing conditions will completelydisassociate duplexes bound to the support. These conditions includesolutions containing kaotropic agents such as urea either alone or incombination with other denaturants such as formamide used either atambient or elevated temperature. The covalent attachment between thefirst and second polynucleotide probes and the bonding of one of theprobes to a surface, however, will be unaffected. Detection of theresulting labelled material bound to the support will indicate thepresence of the target nucleotide sequence in the sample.

Detection of the signal will depend upon the nature of the signalproducing system utilized. If the label or reporter group is an enzyme,additional members of the signal producing system would include enzymesubstrates and so forth. The product of the enzyme reaction ispreferably a luminescent product, or a fluorescent or non-fluorescentdye, any of which can be detected spectrophotometrically, or a productthat can be detected by other spectrometric or electrometric means. Ifthe label is a fluorescent molecule the medium can be irradiated and thefluorescence determined. Where the label is a radioactive group, themedium can be counted to determine the radioactive count.

Another aspect of a method for detecting the presence of apolynucleotide analyte in a sample suspected of containing the analyteinvolves ligating third and fourth sequences of a polynucleotide probe,where the third and fourth sequences are hybridizable to separateportions of the analyte and ligatable, or capable of being renderedligatable, only in the presence of the analyte. When the third andfourth sequences are ligated, the thus formed single strandedpolynucleotide can be incorporated into a circular polynucleotidestrand. The polynucleotide probe further comprises at least partiallycomplementary first and second sequences.

An extension of a polynucleotide primer is formed in the presence ofnucleoside triphosphates and template dependent polynucleotidepolymerase. At least the 3'-end of the polynucleotide primer canhybridize with the first or second sequences of the polynucleotideprobe. Next, extended polynucleotide primer and/or a sequence identicalto and/or complementary with at least that portion of the ligated thirdor fourth sequences complementary to the separate portions of saidanalyte are detected. The presence of any one of the above indicates thepresence of polynucleotide analyte in the sample.

Various techniques can be employed for preparing a polynucleotideprimer, first and second polynucleotide probes, or a single strandedpolynucleotide sequence in accordance with the present invention. Theycan be obtained by biological synthesis or by chemical synthesis. Forshort sequences (up to about 100 nucleotides) chemical synthesis willfrequently be more economical as compared to the biological synthesis.In addition to economy, chemical synthesis provides a convenient way ofincorporating low molecular weight compounds and/or modified basesduring the synthesis step. Furthermore, chemical synthesis is veryflexible in the choice of length and region of the target polynucleotidebinding sequence. The polynucleotide primer can be synthesized bystandard methods such as those used in commercial automated nucleic acidsynthesizers. Chemical synthesis of DNA on a suitably modified glass orresin can result in DNA covalently attached to the surface. This mayoffer advantages in washing and sample handling. For longer sequencesstandard replication methods employed in molecular biology can be usedsuch as those employed in commercial kits for preparation of RNA (e.g.from Promega) and by the use of M13 for single stranded DNA as describedby J. Messing (1983) Methods Enzymol, 101, 20-78.

Other methods of oligonucleotide synthesis include phosphotriester andphosphodiester methods (Narang, et al. (1979) Meth. Enzymol 68: 90) andsynthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters 22:1859-1862) as well as phosphoramidate technique, Caruthers, M. H., etal., "Methods in Enzymology," Vol. 154, pp. 287-314 (1988), and othersdescribed in "Synthesis and Applications of DNA and RNA," S. A. Narang,editor, Academic Press, New York, 1987, and the references containedtherein.

The single stranded polynucleotide or the first and secondpolynucleotide probes can be prepared by enzymatic ligation. Twoappropriate complementary oligonucleotides can be synthesized bystandard automated techniques. They are then enzymatically ligated toanother polynucleotide sequence, for example, by T4 ligase, to producethe single stranded polynucleotide or each individually ligated todifferent polynucleotide sequences, the latter each being hybridizablewith a portion of a polynucleotide analyte. Desired products can then beisolated, for example, by polyacrylamide gel electrophoresis or highperformance liquid chromatography (HPLC).

In some instances, the 3'-end of the single stranded polynucleotide orof the second polynucleotide probe will be modified to prevent reactionwith template dependent DNA polymerase or to append a binding sequence.The 3'-end can, for example, be modified by ligation of adideoxynucleotide or a ribonucleotide followed by oxidation of theribose with periodate followed by reductive amination of the resultingdialdehyde with borohydride and a bulky amine such as aminodextran.

The polynucleotide primer can be prepared by standard automatedtechniques.

As a matter of convenience, the reagents employed in the presentinvention can be provided in a kit in packaged combination withpredetermined amounts of reagents for use in the present method. Inassaying for a polynucleotide analyte in a sample, a kit useful in thepresent method can comprise in packaged combination with other reagentsa polynucleotide primer and first and second polynucleotide probes,which can be labeled or one of which can be bound to a support or can beprovided with groups to render the sequence labeled or bound to asupport. For use in a method of producing multiple copies of a singlestranded polynucleotide, the kit will contain a polynucleotide primer.Either of the kits above can further include in the packaged combinationnucleoside triphosphates such as deoxynucleoside triphosphates, e.g.,deoxyadenosine triphosphate (dATP), deoxyguanosine triphosphate (dGTP),deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate(dTTP). The kit can further include a polynucleotide polymerase and alsomeans for covalently attaching the first and second sequences, such as aligase, and members of a signal producing system.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents which substantiallyoptimize the reactions that need to occur during the present method andto further substantially optimize the sensitivity of the assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay inaccordance with the present invention. Each reagent can be packaged inseparate containers or some reagents can be combined in one containerwhere reactivity and shelf life will permit.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples.

Example 1 Single primer polynucleotide amplification of asingle-stranded polynucleotide with flanking first and secondcomplementary sequences (Oligomer 1)

Oligodeoxyribonucleotide sequences 1, 2, 3, 4, 5, 6, 7, 8 & 9:

Single-stranded polynucleotide with first and second flanking sequences:

Oligomer 1

5' CAA TTA CAC AAG CTT AAT ACA TTC CTT CGA GCT CGG TAC CCG GGG ATC CTCTAG AGT CGA CCT GCA GGC ATG CAA GGA ATG TAT TAA GCT TGT GTA ATT G 3'

First polynucleotide probe A;

Oligomer 2

g' CAA TTA CAC AAG CTT AAT ACA TTC CTT CGA GCT CGG TAC CCG GGG ATC CT3'

First polynucleotide probe B;

Oligomer 3

5' CAA TTA CAC AAG CTT AAT ACA TTC CTT CGA GCT CGG TAC CCG GGG ATC C 3'

Second polynucleotide probe;

Oligomer 4

5' CTA GAG TCG ACC TGC AGG CAT GCA AGG AAT GTA TTA AGC TTG TGT MAT TG 3'

Polynucleotide primer;

Oligomer 5

5' CAA TTA CAC AAG CTT AAT ACA TTC C 3'

Oligomer 6

5' CCG GGG ATC CTC TAG AGT CGA CC 3'

Oligomer 7

5' CCG GGG ATC CCT AGA GTC GAC C 3'

Oligomer 8

5' TCT AGA GTC GAC CTG CAG GCA TGC A 3'

Oligomer 9

5' TGC ATG CCT GCA GGT CGA CTC TAG A 3'

were synthesized by the phosphoramidite method and purified ondenaturing polyacrylamide gels. The 27 5' terminal bases and the 27 3'terminal bases of the 100 mer oligomer 1 are self-complementary andtherefore this molecule will form a "hairpin" or stem-loop structure.Oligomers 2 and 4 represent the 5' 50 bases and 3' 50 bases of oligomer1, respectively. Oligomer 5 is complementary to the 3'terminal 25 basesof oligomers 1 and 4. Oligomers 6 (7) will hybridize to the central 23(22) bases of oligomer 1, but oligomer 7 has a single base deletion (athymidine residue). Oligomers 8 and 9are complementary; oligomer 8 isidentical to oligomer 1 from bases 50 to 74 of oligomer 1, inclusive.

A protocol for DNA amplification of oligomer 1 using oligomer 5 as thepolynucleotide primer is described; variations of this protocol will bedescribed in later examples. One picomole (pmole) of oligomer 1 and 60pmoles of oligomer 5 are combined in a buffer of 50 mM Tris-HCl (pH 8.4@ 25° C.), 50 mM KCl, 2.5 mM Mg Cl₂, 0.2 mg/mL gelatin with 10 nanomoles(nmoles) each dNTP. Five units of Taq polymerase enzyme is added for afinal volume of 50 microliters. Temperature cycling of 94° C. (1minute), 37° C. (2 minutes), and 72° C. (3 minutes) is performed using aprogrammable thermal cycler (Ericomp, Inc.) for a number of cyclesthrough the 3 temperatures. Aliquots from these reactions are withdrawnat various stages and dot-blotted onto GeneScreen Plus® nylon membranes(Du Pont) using protocols recommended by the manufacturer.

The dried membranes are prehybridized for 3 hours at 65° C. in asolution of 0.75M NaCl, 0.15M Tris-HCl (pH8.0), 10 mM EDTA, 5×Denhardt's solution (1 g Ficoll, 1 g polyvinyl pyrrolidone, 1 g BSA in atotal vol 1000 ml H₂ O), 20 mM sodium phosphate, 250 mg/mL sheareddenatured calf thymus DNA, and 0.5% SDS. In order to assay for theformation of amplified oligomer 1, ³² P5' end-labeledoligodeoxynucleotide probe (˜10' DPM, 100-1000 Ci/mmole) is added withfresh prehybridization solution and hybridized overnight with gentleagitation at 60° C. The hybridized membranes are typically washed in abuffer of 0.3M NaCl, 0.06M Tris-HCl (pH 8.0), 4 mM EDTA, and 0.5% SDS at60° C. for 30 minutes with one change of wash buffer. Washed membranesare exposed to Kodak X-Omat® AR film for varying lengths of time,sometimes with a single intensifying screen (Du Pont Cronex®Lightning-Plus).

FIG. 1 shows the results of amplification of 1 pmole of oligomer 1 inthe presence of 60 pmoles of polynucleotide primer, oligomer 5. Theprobe in this case is oligomer 5 and the standards (Lane A, 1-10) arevarying amounts of oligomer 1. No detectable hybridization occurs ifoligomer 5 is left out of the amplification reaction. A small amount ofamplification of oligomer 5 is detected when oligomer 1 is not added.

This blot was stripped of radioactive probe by washing for 10 minutes atroom temperature in 0.4N NaOH and neutralizing in 1M Tris-HCl (pH 7.5),0.3M NaCl, 4 mM EDTA. The membrane was then hybridized to 5'³² P-labeledoligomer 9 (see FIG. 2). In this case, amplification is detected in thereaction containing both oligomers 1 and 5 and not in those containingeither 1 or 5 separately.

FIG. 1 Dot-blot hybridization of ³² P oligomer 5 to single primerpolynucleotide amplification products

Row A. Lanes 1-10--Standards of 1.8, 0.9, 0.454, 0.18, 0.09, 0.045,0.018, 0.009, 0.0045, 0.0018 nmoles oligomer #1.

Row A. Lane 11--Negative control of 1 μg sheared, denatured calf thymusDNA.

Row B. Lanes 7-11--Five μL aliquots from the 10, 20, 30, 40, and 50thtemperature cycle of amplification reaction containing 60 pmoles ofoligomer 5 with no oligomer 1. Note some hybridization of products fromcycle 50 to the oligomer 5 probe.

Row C. Lanes 1-5--5 μL aliquots from the 10, 20, 30, 40 and 50th cyclecontaining oligomer 1 only (1 pmole). Note absence of detectableamplification product.

Row C. Lanes 7-11--5 μL aliquots from the 10, 20, 30, 40, and 50th cyclecontaining both oligomers 1 and 5. Amplification exceeds 2000-fold (1pmole to >1.8 nmoles).

FIG. 2 Dot-blot hybridization of ³² P oligomer 9 to single primerpolynucleotide amplification products

The blot in FIG. 1 was stripped of hybridized ³² P oligomer 5 andhybridized to ³² P oligomer 9 probe. Results are essentially as in FIG.1 except that hybridization in row B, lanes 7-11, cannot be detectedwith this probe.

Example 2 Single primer polynucleotide amplification of cloned DNA fromNeisseria gonorrhoeae Oligodeoxyribonucleotide sequences 1, 2, 3, 4, 5:

Single-stranded polynucleotide target;

Oligomer 1

5'ACT TGG GCT ATC ACT TCC CTG AAC CGC GTG CTT TTA CTA ATA GAG AAC GAGCAA GGC TTC AAA GTT TTC CTG ATG ATT TTG AGT TTG TCG GAT CAA CAA CTG AAGT 3'

First polynucleotide substrate;

Oligomer 2

5' ACG GTT CCA TCA AAA GGG GGG AAT TCA CTT TTC TCT ATC ACT GAT AGG GAGTGG TM AAT AAC TCT ATC AAT GAT AGA GAC TTC AGT TGT TGA TCC GAC TTC GAAMGAG C

Second polynucleotide substrate;

Oligomer 3

5' ACG GTT CCA TCA AAA GGG GGG AAT TCT GTG TGG AAT TGT GAG CGG ATA ACAATT TCA CAC AAC TTG GGC TAT CAC TTC CCT GGA TCC CCA T 3'

Polynucleotide primer;

Oligomer 4

5' ACG GTT CCA TCA AAA GGG GG

Polynucleotide probe;

Oligomer 5

5' TM TAG AGA ACG AGC AAG GCT TCA A 3'

Oligomer 1 is one strand of a double-stranged Rsa I restriction fragmentfrom a N. gonorrhoeae (N. g.) genomic clone. The total cloned DNA insertrepresents a 7 kilobase (Kb) Sau 3AI restriction fragment originatingfrom N. g. strain 125. This 7 Kb fragment was inserted into Bam HIlinearized vector pGEM 3Zf(+) from Promega Biotech.

The 3'terminal twenty base of oligomer 1 hybridizes to bases number80-99, inclusive, of oligomer 2 (see FIG. 8, A). This forms a primedsubstrate for DNA polymerization upon the oligomer 2 template from the3' terminus of oligomer 1, catalyzed by Taq polymerase. The 185-baselong product of this polymerization, when rendered single-stranded bythe thermal denaturation, base pairs with its 3' terminal twenty basesto oligomer 4 (FIG. 8, B). This is a substrate for Taq polymerase whichpolymerizes upon the primed 185-base template to form a complementarystrand product of 185 bases.

The denatured product of the preceding polymerization has twenty basesat its 3' terminus which hybridizes to oligomer 3 at nucleotides 62-81,inclusive, and therefore is a substrate for Taq polymerase to extend 61bases upon the oligomer 3 template (FIG. 8, C). The product, whenthermally denatured, produced 246-base long polynucleotide 6 of Scheme5.

Polynucleotide 6 forms a stem-loop structure with a stem of 20 basepairs of complementary sequence at the 5' and 3' termini. It followsthat the complement of 6 also forms a stem-loop structure. Both 6 andits complement hybridizes to primer oligomer 4 at their respective 3'terminus, providing a substrate for Taq polymerase and thereby allowingsingle primer polynucleotide amplification of the type described inExample 1. To summarize, the presence of oligomer 1 has allowed, byalternative polymerase extension of primed templates and thermaldenaturation, the "linking" of sequences complementary to oligomers 2and 3 forming polynucleotide 6.

The following experiment is an example of this technology. Five assayswere performed containing the following components:

    __________________________________________________________________________            dATP                                                                          dCTP                                                                          dGTP                    Rsa I Digest                                                                         5 Units                                Assay                                                                             10 X                                                                              dTTP Oligomer                                                                           Oligomer                                                                             Oligomer                                                                             of N. q.                                                                             TAQ                                    No. Buffer                                                                            (nmoles)                                                                           4    2      3      Genomic Clone                                                                        Polymerase                             __________________________________________________________________________    1   +   100 ea.                                                                            18 pmoles                                                                          20 pmoles                                                                            20 moles                                                                             None   +                                      2   +   100 ea.                                                                            18 pmoles                                                                          20 pmoles                                                                            20 pmoles                                                                            25 fmoles                                                                            +                                      3   +   100 ea.                                                                            18 pmoles                                                                          200                                                                              fmoles                                                                            200                                                                              fmoles                                                                            25 fmoles                                                                            +                                      4   +   100 ea.                                                                            18 pmoles                                                                          2  fmoles                                                                            2  fmoles                                                                            25 fmoles                                                                            +                                      5   +   100 ea.                                                                            18 pmoles                                                                          20 pmoles                                                                            20 pmoles                                                                            25 amoles                                                                            +                                      __________________________________________________________________________

Ten times (10×) buffer is 0.1M Tris-HCl, pH 8.4 (at 25° C.), 0.5M KC1,60 mM MgCl₂, 2 mg/mL gelatin. Final volume of assay was 100 μL. Allcomponents, except Taq polymerase, were added to eppendorf tubes,overlaid with 50 μL mineral oil and heated 95° C. for 3 minutes. Tubeswere then allowed to slow cool over 30 minutes to less than 50° C. Fiveunites of Taq polymerase were then added and thermal cycling was begunin an ERICOMP thermal block: 94° C. for 30 sec, 50° C. for 30 sec, 72°C. for 2 min, then repetition of this three-temperature regimen (1cycle). Ten microliter aliquots of these five assays were withdrawn at20, 40, and 60 cycles and frozen.

Five microliters of the frozen aliquots were thawed and analyzed bydot-blotting onto Genescreen Plus™ nylon membranes (DuPont) usingprotocols recommended by the manufacturer. The blots were processed asdescribed in Example 1. The ³² P-kinased probe oligomer 5 (100-1000Ci/mmole, ˜10⁷ dpm) was used to hybridize to the dot blot. Oligomer 5 isidentical to bases 38-62, inclusive, of the target oligomer 1.

The blot in FIG. 3 demonstrates that by 60 temperature cycles, allassays containing target DNA had greater than 25 fmoles/5 μL of DNAhybridizable to the probe. In dot blot 5E, this represents anamplification of 5×10⁻¹⁸ moles hybridizable sequence to >25×10⁻¹⁵ moleshybridizable sequence, or about a 5×10³ -fold amplification.

FIG. 3 Legend Dot-blot Hybridization of ³² P Oligomer 5 to AmplificationProducts of Assays 1-5

Row A. Lanes 1-4--Standards of 25, 2.5, 0.25, and 0.025fmoles N.gonorrhoeae genomic clone.

Row C. Lanes 1-5Five microliter aliquots of 20th temperature cycle ofassays 1-5, respectively.

Row D. Lanes 1-5--Five microliter aliquots of 40th temperature cycle ofassays 1-5, respectively.

Row E. Lanes 1-5--Five microliter aliquots of 60th temperature cycle ofassays 1-5, respectively.

What is claimed is:
 1. A kit for use in determining a polynucleotideanalyte, which comprises in packaged combination:a first polynucleotideprobe having a first nucleotide sequence capable of hybridizing to afirst portion of said polynucleotide analyte, a second polynucleotideprobe having a second nucleotide sequence capable of hybridizing to asecond portion of the same strand of said polynucleotide analyte otherthan with the portion recognized by said first nucleotide sequence ofsaid first polynucleotide probe, said first polynucleotide probe andsaid second polynucleotide probe each comprising a sequence ofnucleotides that are hybridizable with one another when said firstnucleotide sequence and said second nucleotide sequence are hybridizedto their respective portions of said polynucleotide analyte, each saidsequence of nucleotides being located in its respective polynucleotideprobe at or near the end thereof opposite the site of said firstnucleotide sequence or said second nucleotide sequence, respectively,means for covalently attaching said first and second polynucleotidesequences when said first and second polynucleotide probes arehybridized with said polynucleotide analyte thereby forming covalentlyattached first and second polynucleotide sequences such that apolynucleotide primer that hybridizes to and is extended along saidcovalently attached sequences forms an extension product comprising saidfirst and second polynucleotide sequences, a polynucleotide primercapable of hybridizing with said second polynucleotide probe and ofbeing extended along said covalently attached sequences in the directionof said first polynucleotide probe, and means for extending saidpolynucleotide primer, said means comprising a polynucleotide polymeraseand one or more deoxynucleoside triphosphates.
 2. The kit of claim 1further comprising:a ligase as a means for covalently attaching saidfirst and second nucleotide sequences.
 3. The kit of claim 1 whereinsaid first polynucleotide probe has, of having, means for immobilizingsaid probe.
 4. The kit of claim 1 wherein at least one of said first orsecond probe contains a polynucleotide sequence that serves as a label.5. The kit of claim 1 wherein said first and second portions of saidpolynucleotide analyte are not contiguous.